Applications of nanoparticles in plant genetic transformation and crop improvement

Introduction

Nanotechnology has emerged as a transformative and interdisciplinary field with farreaching implications for genetic engineering, biotechnology, and agricultural sciences. The conceptual foundation of nanotechnology was first articulated by physicist Richard P. Feynman in his landmark lecture "There's Plenty of Room at the Bottom", in which he envisioned the precise manipulation of matter at atomic and molecular scales (Feynman, 1959) [13] . The term nanotechnology was later coined by Norio Taniguchi in 1974 [26] to describe precision engineering and material processing at the nanometre scale (Taniguchi, 1974) [26] . According to the National Nanotechnology Initiative (NNI), nanotechnology involves the understanding and manipulation of matter with at least one dimension in the size range of 1-100 nanometres. Similarly, the European Commission defines nanotechnology as the science and technology of materials and systems that exhibit novel properties and functions due to their nanoscale dimensions (European Commission, 2011) [12] . At this scale, materials display size-dependent physical, chemical, and biological properties that are markedly different from their bulk counterparts, enabling innovative applications across multiple disciplines. Nanotechnology has rapidly expanded into diverse fields, including medicine, molecular and synthetic biology, biochemistry, agriculture, environmental science, and information and communication technologies. The unique behavior of nanomaterials arises primarily from their high surface area-to-volume ratio and the dominance of surface atoms, which significantly alter surface energy, chemical reactivity, mechanical strength, optical behavior, and electrical conductivity (Guston, 2007; Bhushan, 2017) [15,4] . Unlike bulk materials that exhibit relatively constant properties, nanoscale materials demonstrate enhanced reactivity and tunable functional characteristics, making them particularly attractive for biological applications. In addition to surface effects, quantum confinement phenomena at the nanoscale impart distinct optical, magnetic, and electronic properties that are absent in larger materials (Bhushan, 2017) [4] . These quantum effects enable nanoparticles to interact efficiently with biomolecules such as DNA, RNA, proteins, and lipids, thereby facilitating their application in gene delivery, imaging, sensing, and molecular diagnostics. Based on composition and structural characteristics, nanomaterials are broadly classified into metal nanoparticles, metal oxide nanoparticles, lipid-and liposome-based nanoparticles, polymer-based nanoparticles, dendrimers, and silica-and carbon-based nanomaterials. Due to their physicochemical tunability, biocompatibility, and functional versatility, these nanomaterials have gained increasing attention in plant biotechnology, particularly for genetic transformation, targeted gene delivery, and crop improvement.

In the context of agriculture, nanotechnology offers promising tools for overcoming limitations associated with conventional breeding and transformation methods. Nanoparticle-mediated delivery systems enable efficient transport of genetic material across plant cellular barriers, including the cell wall, and provide opportunities for precise genetic modification. Consequently, nanotechnology holds significant potential for accelerating the development of climate-resilient crops with enhanced resistance to biotic and abiotic stresses, improved nutrient use efficiency, and sustainable yield stability.

Nanobiotechnology and Its Applications in Agriculture

Nanobiotechnology represents the convergence of nanotechnology and biological sciences, enabling the application and address complex challenges in agriculture, and genetic engineering by enhancing precision, efficiency, and sustainability offers innovative strategies to improve crop productivity, stress tolerance, and resource-use efficiency under changing climatic conditions. A wide range of nanoparticles is employed in nanobiotechnological applications, including metal and metal oxide nanoparticles, clay-based nanomaterials, solid lipid nanoparticles, natural polymer-based nanoparticles, and synthetic dendrimers, which can positively influence plant growth and development by enhancing seed germination, improving nutrient uptake, regulating phytohormone activity, and promoting overall plant health (Barrena et al., 2009) [3] . These effects are largely attributed to the ability of nanoparticles to interact efficiently with plant tissues and cellular components. Recent advances in plant nanobiotechnology highlight the use of nanoparticles as nanocarriers for the targeted delivery of agrochemicals, including fertilizers, herbicides, pesticides, plant growth regulators, and bioactive molecules. Nanocarrier-based delivery systems enable controlled and site-specific release of active compounds, thereby enhancing efficacy while minimizing dosage requirements, off-target effects, and environmental contamination. Such precision agriculture approaches contribute to sustainable crop management and reduced chemical inputs. Among various nanomaterials, gold nanoparticles (AuNPs) are among the most extensively studied for biomolecule delivery due to their excellent biocompatibility, low cytotoxicity, and tuneable surface chemistry. AuNPs have been successfully employed for the delivery of proteins and nucleic acids in both plant and animal systems. They can bind and protect DNA from enzymatic degradation and facilitate controlled release of genetic cargo through redoxresponsive mechanisms, such as glutathione-mediated dissociation (Han et al., 200) [16] . Nanoparticle-mediated gene delivery has emerged as an alternative to Agrobacterium and biolistic methods. Carbon nanotubes and mesoporous silica nanoparticles have enabled DNA, RNA, and CRISPR-Cas components delivery into plant cells without genome integration or tissue damage (Demirer et al., 2019; Cunningham et al., 2018) [8,5] . Recent studies suggest that nanotechnology-enabled approaches can accelerate the development of climate-resilient crops by improving tolerance to abiotic stresses such as drought, heat, salinity, and heavy metal toxicity through enhanced antioxidant defense and stress-responsive gene regulation (Dimkpa and Bindraban, 2018) [10] . By [14,9] .  Gold nanoparticles (AuNPs) have been successfully used for DNA and protein delivery in plants, protecting nucleic acids from degradation and enabling controlled release, thereby improving transformation efficiency (Pissuwan et al., 2011) [22] .  Clay nanosheets and layered double hydroxides (LDHs) have been developed as carriers for double-stranded RNA (dsRNA), enabling spray-induced gene silencing (SIGS) for viral and fungal disease control, offering a non-transgenic plant protection strategy (Mitter et al., 2017) [19] .  Zinc oxide (ZnO) and silicon nanoparticles have been shown to enhance seed germination, nutrient uptake, photosynthetic efficiency, and antioxidant enzyme activity, leading to improved plant growth and stress tolerance under drought and salinity conditions (Rastogi et al., 2017) [24] .  Nano fertilizers based on iron, zinc, nitrogen, and phosphorus nanoparticles have demonstrated higher nutrient-use efficiency, reduced leaching losses, and sustained nutrient release compared to conventional fertilizers, contributing to sustainable agriculture (DeRosa et al., 2010; Kah et al., 2019) [7,17] .  Silver and metal oxide nanoparticles exhibit strong antimicrobial activity against plant pathogens, reducing disease incidence and post-harvest losses, although their dosage-dependent phytotoxicity requires careful evaluation (Rai et al., 2012) [23] .

 Polymeric and chitosan-based nanoparticles have been widely explored for delivery of pesticides, plant growth regulators, and nucleic acids, owing to their biodegradability, low toxicity, and enhanced plant compatibility.  Magnetic nanoparticles have been applied for targeted delivery of biomolecules and for tracking nanoparticle movement within plant tissues, opening new avenues for precision agriculture and plant imaging (Tian et al., 2013) [28] .

Nanomaterial-Driven Genetic Transformation in Plants

This approach combines advances in nanomaterial engineering with plant biotechnology to enable efficient, precise, and minimally invasive delivery of nucleic acids and genome-editing components into plant cells. The workflow describes the systematic steps involved in nanomaterial-mediated transformation, including nanoparticle synthesis and characterization, surface functionalization and cargo loading, preparation of plant tissues, nanomaterial-assisted delivery, intracellular release and expression of genetic material, and subsequent validation of transformation efficiency and biosafety. Collectively, this framework highlights the potential of nanomaterial-driven strategies as a robust alternative to conventional plant transformation methods for sustainable crop improvement.

 Synthesis and Characterization of Nanoparticles:

Nanoparticles (e.g., carbon nanotubes, gold, silica, magnetic, polymeric, or clay-based) are synthesized using chemical or biological methods. Physicochemical properties such as size, shape, surface charge, dispersity, and stability are characterized using techniques including transmission/scanning electron microscopy (TEM/SEM), dynamic light scattering (DLS), and zeta potential analysis (Bhushan, 2017; Cunningham et al., 2018) [4,5] . [16,22]  Plant Material Preparation: Target plant material such as leaves, embryos, callus, meristems, or protoplasts is prepared under sterile conditions. In some approaches, intact plants or seedlings are used to enable tissue culture-independent delivery (Giraldo et al., 2014; Cunningham et al., 2018) [14,5] [8,9] .

 Validation of Transformation and Gene Expression:

Successful delivery and expression are confirmed using molecular assays such as PCR, qRT-PCR, reporter gene analysis (e.g., GFP, GUS), Western blotting, or sequencing for genome edits. Phenotypic and physiological responses are evaluated where applicable (Torney et al., 2007; Martin-Ortigosa et al., 2012) [29,20] .  Assessment of Efficiency and Biosafety: Transformation efficiency, tissue viability, and potential nanoparticle toxicity are evaluated. Comparative analysis with conventional transformation methods is performed to assess efficiency, reproducibility, and environmental safety (Mitter et al., 2017; Kah et al., 2019) [19,17] . [29] . Calcium phosphate nanoparticles have shown higher transformation efficiency than Agrobacterium tumefaciens and enabled successful delivery of GFP-encoding plasmids into tobacco cells (Ardekani et al., 2014) [2] .

Metal-based nanoparticles have also been explored as gene delivery vehicles. Carbon-supported gold nanoparticles improved DNA delivery efficiency, reduced cellular damage, and enhanced regeneration and transformation frequency in multiple plant species compared to conventional gold particles used in gene gun systems (Vijayakumar et al., 2010) [30] . Magnetic gold nanoparticles and gold nanoparticle-based biolistics have further enabled effective plasmid delivery and stable transformation in crops such as canola, carrot, and rice (Chang et al., 2013) [6] . More recently, silver nanoparticles have emerged as cost-effective microcarriers, significantly increasing transformation efficiency while drastically reducing material costs (Rajkumari et al., 2021) [25] .

Conclusion

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